Immunological diversity
Gilbert Chu
Advanced Immunology (I), January 2004
I. Antibody diversity: historical roots
430 BC Thucydides
On bubonic plague: "It was with those who had recovered
from disease that the sick and the dying found most
compassion. These knew what it was from experience, and
had now no fear themselves; for the same man was never
attacked twice - never at least fatally."
1796
Jenner
Noted that cowpox was rarely followed by smallpox.
Showed that cowpox innoculum protected from smallpox
Pasteur coined “vaccine” from vacca, cow in Latin.
1901
Landsteiner et al.
Discovered antibodies against ABO blood antigens.
Made antibodies against many organic molecules: specificity
and diversity.
Discovered antibodies against the red blood cell antigen in
paroxysmal cold hemoglobinuria: autoimmunity
How are antibodies of virtually unlimited diversity generated?
II. Immunoglobulins
A. The antibody molecule
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B. Immunoglobulin gene
H chain locus (Chr 12)
V1-V500
D1-D12
J1-J4
C
C
C3
C1
C2b C2a
C
C
 chain locus (Chr 6)
V1-V250
J1-J5
C
 chain locus (Chr 16)
V2
J2 C2
V1
J3 C3
J1 C1
C. Mechanisms for generating antibody diversity
1. V(D)J recombination
2. Somatic hypermutation
3. Class switch recombination
III. V(D)J recombination
A. Recombination signal sequences (RSS) direct V(D)J recombination
1. RSS are located adjacent to the V, D, and J exons of the immunoglobulin genes
2. Each RSS consists of a conserved heptamer separated from a conserved nonamer by
a nonconserved spacer of 12 or 23 nucleotides.
3. "The 12/23 rule": recombination only occurs between a RSS containing a 12 spacer
and
a RSS containing a 23 spacer.
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B. V(D)J recombination involves cleavage and rejoining
1. Cleavage is initiated by RAG1/RAG2 (recombination activating genes)
a. Binding to RSS
RAG1 binds to nonamer in the RSS; binding is stabilized by the heptamer
RAG2 is recruited to the RSS (more efficiently to the 12-RSS)
b. Synapsis of 12-RSS and 23-RSS
c. Coordinated cleavage by the 12/23 rule
cleavage is initiated by nick adjacent to heptamer
cleavage is completed by formation of hairpin coding end and blunt signal end
C. DNA ends are modified by addition and deletion
1. N-nucleotide addition by terminal deoxynucleotidyl transferase (TdT), a templateindependent DNA polymerase expressed only in lymphoid cells
2. P-nucleotide addition by asymmetric opening of hairpin coding ends
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3. Nucleotide deletion
D. DNA pathways in V(D)J recombination
E. Evolution of V(D)J recombination
1. RAG1 and RAG2 contain no introns and are tightly linked on the same chromosome
2. RAG1 and RAG2 are conserved back to the evolution of the jawless fish (e.g., hagfish
and lamprey)
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3. Evolutionary hypothesis: a transposon carrying RAG1/RAG2 and associated RSSs
infected a precursor jawed fish
4. Subsequent gene duplication created the fragmented immunoglobulin locus
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F. Mutant cell lines: defective for both V(D)J recombination and double-strand break repair
1. Scid mouse (group 7)
a. mouse with severe combined immunodeficiency lacking mature Band T cells
b. defective in the joining of coding ends; normal in the joining of signal ends
c. hypersensitive to ionizing radiation
d. the scid mouse suggested a link between V(D)J recombination and the repair of
DNA double-strand breaks
2. Mutant nonlymphoid cells can be tested for V(D)J recombination
a. Mutagenesis of Chinese hamster epithelial cells generated several X-ray sensitive cell
lines
b. These cells were co-transfected with RAG1, RAG2, and V(D)J recombination substrates
c. The cells were then assayed for either coding joint formation or signal joint formation
3. Cells from groups 4 and 5 are defective in coding and signal joint formation
4. Conclusion: a common non-homologous end-joining pathway repairs the doublestrand breaks created by V(D)J recombination or ionizing radiation.
G. Proteins involved in non-homologous end-joining
Protein
Enzymatic activity
Ku
DNA-PKcs
DNA end-binding
DNA-dependent protein kinase: activated by DNA
ends
DNA ligase
exonuclease
exo/endonuclease
XRCC4 / ligase IV
Artemis
Rad50/ Mre11/ Nbs1
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H. Human diseases with defects in non-homologous end-joining
1. Severe combined immunodeficiency with radiation sensitivity (Artemis)
2. Ataxia telangiectasia-like disorder (Mre11)
3. Nijmegan breakage syndrome (Nbs1)
I. Roles for Ku and DNA-PKcs during non-homologous end-joining
1. Ku recruits DNA-PKcs to DNA ends
2. DNA-PKcs brings the DNA ends together in a synaptic complex
3. The kinase is activated after the DNA ends are brought together
Model for activation of DNA-PKcs by DNA
4. Proteins with nuclease and/or polymerase activities process the DNA ends
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5. Autophosphorylation of DNA-PKcs induces it to fall off the DNA
6. The ends are ligated together
J. Questions about non-homologous end-joining
1. Protein questions
a. What are the DNA polymerases?
b. What are the nucleases?
2. Phosphorylation questions
a. Which proteins are targeted by DNA-PK?
b. How does phosphorylation regulate these proteins?
c. How does DNA-PK phosphorylate these proteins before phosphorylating itself?
IV. Somatic hypermutation (SHM)
A. SHM targets immunoglobulin genes (but not T cell receptor genes)
B. Hypermutation requires active transcription, and involves DNA single-strand breaks
C. Activation-induced deaminase (AID)
1. Expressed only in activated B cells
2. Converts C to U in single-stranded DNA
D. Model for somatic hypermutation
1. AID converts C to U on nontranscribed
strand
2. Uracil DNA glycosylase removes the U,
leaving an apurinic site in the DNA
3. AP (apurinic/apyrimidinic) endonuclease
nicks the DNA adjacent to the apurinic site
4. Exonuclease removes the apurinic ribose
5. An error-prone DNA polymerase fills the gap
6. How is C mutated on both strands with the
same frequency? How does SHM target the
Ig locus, but not other loci?
V. Class switch recombination (CSR)
A. CSR rearranges the constant regions to generate different antibody isotypes
1. CSR regions are located 5' to each CH gene, except for C
2. CSR regions consist of repeats of GAGCT and GGGGGT.
For example,  switch region is [(GAGCT)nGGGGGT]150.
B. CSR requires active transcription
C. CSR occurs via DNA double-strand breaks
1. Requires Ku and DNA-PKcs
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2. CSR junctions show characteristics of non-homologous end-joining



a. Deletions to regions of microhomology
b. Duplications from DNA polymerase activity
D. Model for class switch recombination
How does AID initiate CSR at one locus and SHM at another?
(The C-terminus of AID is required for CSR but not SHM.)
VI. Summary
A. Diversity is generated by multiple mechanisms
1. V(D)J recombination
2. Somatic hypermutation
3. Class switch recombination
B. Some components are lymphocyte-specific: RAG1/RAG2, TdT, AID
C. General components are involved in double-strand break repair and base excision repair
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References
1. Abbas AK, Lichtman AH, Pober JS (1991) Cellular and Molecular Immunology. WB
Saunders Co., Philadelphia, PA.
2. Oettinger M, Schatz D, Gorka D, Baltimore D (1990) RAG-1 and RAG-2, adjacent genes
that synergistically activate V(D)J recombination. Science 248: 1517-1523.
4. McBlane JF et al. (1995) Cleavage at a V(D)J recombination signal requires only RAG1
and RAG2 proteins and occurs in two steps. Cell 83: 387-395.
5. Agrawal A, Eastman Q, Schatz D (1998) Transposition mediated by RAG1 and RAG2 and
its implications for the evolution of the immune system. Nature 394: 744-751.
6. Hiom K, Melek M, Gellert M (1998) DNA transpostion by the RAG1 and RAG2 proteins:
a possible source of oncogenic translocations. Cell 94: 463-470.
7. Smider V, Rathmell WK, Lieber M, Chu G (1994) Restoration of X-ray resistance and
V(D)J recombination in mutant cells by Ku cDNA. Science 266: 288-291.
8. Chu G (1997) Double-strand break repair. J. Biol. Chem. 272: 24097-24100.
9. Leuther K, Hammarsten O, Kornberg R, Chu G (1999) Structure of DNA-dependent
protein kinase: implications for its regulation by DNA. EMBO J. 18: 1114-1123.
10. Stavnezer J, Bradley S (2002) Does activation-induced deaminase initiate antibody
diversification by DNA deamination? Trends Genet 18: 541-543.
11. Di Noia J, Neuberger M (2002) Altering the pathway of immunoglobulin hypermutation by
inhibiting uracil-DNA glycosylase. Nature 19: 43-48.
12. Casellas R et al. (1998) Ku80 is required for immunoglobulin isotype switching. EMBO J.
17: 2404-2411.
13. Chen X, Kinoshita K, Honjo T (2001) Variable deletion and duplication at recombination
junction ends: implication for staggered double-strand cleavage in class-switch
recombination. Proc Natl Acad Sci USA 98: 13860-13865.
14. Chaudhuri J, Tian M, Khuong C, Chua K, Pinaud E, Alt F (2003) Transcription-targeted
DNA deamination by the AID antibody diversification enzyme. Nature 422: 726-730.
15. Pham P, Bransteitter R, Petruska J, Goodman MF (2003) Processive AID-catalyzed
cytosine deamination on single-stranded DNA stimulates somatic hypermutation. Nature
424: 103-107.
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16. Barreto V, Reina-San-Martin B, Ramiro AR, McBride KM, Nussenzweig MC (2003) Cterminal deletion of AID uncouples class switch recombination from somatic
hypermutation and gene conversion. Mol Cell 12: 501-508.
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Immunoglobulin questions
Gilbert Chu
Advanced Immunology (I), January, 2004
1.
V(D)J recombination is directed by recombination signal sequences (RSS).
A. Draw a diagram showing how RSS can direct a deletion in the genomic DNA.
B. Draw a diagram showing how RSS can direct an inversion in the genomic DNA.
In answering parts A and B, show all of the DNA products.
2.
V(D)J recombination is directed by recombination signal sequences (RSS).
A. How do RSS prevent V to J recombination in the heavy chain locus?
B. Explain how cleavage at the RSS is catalyzed. What are the enzymes? Draw the
DNA intermediates and the recruitment of the enzymes.
3.
The formation of coding joints during V(D)J recombination sometimes involves the
addition of palindromic (P) nucleotides.
A. What is the mechanism for P-nucleotide addition?
B. List the enzymes that would be required for this process.
4.
The formation of coding joints during V(D)J recombination sometimes involves the
addition of random (N) nucleotides.
A. What is the enzymatic mechanism for N-nucleotide addition? Does this mechanism
operate at signal joints? What types of cells does it operate in?
B. How would you use a genetically engineered mouse to determine whether this
enzymatic mechanism accounts for all N-nucleotide addition?
5.
Review the evidence that class switch recombination requires the same non-homologous
end-joining pathway that is used for V(D)J recombination.
6.
AID is required for both somatic hypermutation and class switch recombination.
A. Describe the proposed enzymatic activity for AID
B. Explain how this activity can lead to a DNA nick during class switch recombination.
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Immunoglobulin answers
Gilbert Chu
Advanced Immunology (I), January, 2003
1.
A. Recombination between two RSS in head to head orientation with respect to each
other. There is deletion of that part of the chromosome containing the recombining
RSS and the formation of a circular episome.
B. Recombination between two RSS in head to tail orientation with respect to each
other. There is a chromosomal inversion without deletion.
2.
A. The 12-23 rule. In heavy chain locus, the RSS adjacent to V and J segments have
spacers of 23 nucleotides. The RSS adjacent to the D segments have spacers of 12
nucleotides. Recombination only occurs between RSS with different spacers.
B. RAG1 and RAG2.
RAG1 is recruited to the nonamer. RAG2 is then recruited.
First step: nick adjacent to the RSS leaving a 3' OH at the coding end.
Second step: formation of a hairpin coding end and blunt signal end.
3.
A. Formation of a hairpin, asymmetric cleavage of the hairpin, and end joining.
B. The enzymes: RAG1, RAG2 for cleavage and a presumed hairpin endonuclease.
4.
A. TdT. By some unknown mechanism, it must be prevented from operating at signal
ends, since signal joints are formed without addition or subtraction of nucleotides.
TdT is expressed only in lymphoid cells.
B. Make a mouse knocked out for the TdT gene and analyze coding joints for Nnucleotide addition.
5.
Both processes require Ku and DNA-PK. The junctions formed during both processes
involve deletions back to regions of microhomology.
6.
A. AID acts on DNA as a deoxycytidine deaminase, converting C to U
B. After conversion of C to U, the combined activity of uracil DNA glycosylase and AP
endonuclease produces a nick in the DNA.
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